The developing lens constitutes a powerful system for understanding the molecular basis of tissue development, and it is also the target of the leading cause of blindness, cataract. During the prior grant period, we made major progress in elucidating the regulation of a key lens formation gene, Pax6, via signaling pathways (Notch) and transcription factors (Sox, Pou, Meis). We also developed a bioinformatics method, iSyTE, for efficiently using lens expression data to predict functionally important genes in lens development, and we identified three genes, Pvrl3, Sep15 and Tdrd7, that when mutated cause cataracts. Using Tdrd7 as an entry point, we uncovered a new and important mode of post-transcriptional regulation that operates during lens fiber cell differentiation. Tdrd7 encodes an RNA-binding protein, and is mutated in human congenital cataracts, a phenotype also present in Tdrd7 mouse mutants. Tdrd7 is a component of a novel class of RNA granules (Tdrd7-RGs) that are expressed in developing lens fiber cells, and Tdrd7-RGs bind a specific subset of lens mRNAs and, potentially, either sequester them for translation or target them for degradation. In the former class are mRNAs encoding lens fiber cell components mutated in cataracts, while the latter class includes transcripts such as Pax6 that require down- regulation for fiber cell differentiation. These studies provide mechanistic information about the gene regulatory network (GRN) that controls vertebrate lens development. In this renewal, we propose to further expand our understanding of the lens development GRN by focusing on the role of post-transcriptional regulation by Tdrd7 in lens fiber cell differentiation. We hypothesize that the molecular function of Tdrd7 in Tdrd7-RGs is required for normal lens development. By identifying additional genes in the Tdrd7 functional pathway and additional Tdrd7-RG protein components, we propose to define a Tdrd7 regulatory pathway and to evaluate its overall significance. In Aim 1, we will use conditional and transgenic Tdrd7 mouse alleles, and potentially complementation in mouse and zebrafish to dissect the Tdrd7 function in lens development. For Aim 2, we have focused on a ligand-receptor pair, Ephrin A5 and its receptor Epha2, that when mutated appears to phenocopy the Tdrd7 null mutant. Epha2 mRNA binds Tdrd7, and hence appears to act directly within the Tdrd7 pathway. We will now establish the nature of the regulatory relationship between these signaling molecules and Tdrd7 continue to identify Tdrd7 phenocopy genes, and test whether they encode Tdrd7 pathway components. Lastly, in Aim 3, we will determine how Tdrd7 executes its post-transcriptional function in lens development by biochemically identifying additional RNA and protein Tdrd7-RG components, and by testing whether these mRNAs are dynamically exchanged between different RG types according to their fate. These studies will increase our understanding of lens development, and provide new information on post-transcriptional regulatory mechanisms in oculogenesis.